Biotechnology for Crop Protection - American Chemical Society

Chapter

Genetically

Engineered

31

Viral

Insecticides

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 2, 2018 | https://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch031

Practical Considerations David W. Miller Genetics Institute, Inc., 87 Cambridge Park Drive, Cambridge, MA 02140 Baculoviruses play a central role in the natural control of insect pest populations, chiefly Lepidoptera. This has sustained an interest in the commercial potential of these as larvicides in several pest control situations; however, performance drawbacks have limited their general usefulness. Modification of the viruses through genetic engineering is anticipated to greatly increase their effectiveness. How this may be accomplished w i l l be discussed. The effect modifications may have on the well-established safety of these agents as well as their perceived safety w i l l also be covered. Pertinent examples from our work and that of others are discussed.

The need to find effective alternatives to chemical insecticides has kept alive an interest in the use of naturally occurring insect pathogens as control agents. With the advent of genetic engineering technology, an opportunity has emerged for alleviating the commercial shortcomings of these pathogens and fostering the creation of a new generation of products. Our desire to enter this challenging f i e l d led us to select insect viruses, specifically a baculovirus, as our model system. Their s u i t a b i l i t y to the techniques of genetic engineering and their well-chronicled safety and natural efficacy make them 0097-6156/88/0379-0405$06.00A) © 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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an o b v i o u s c h o i c e . E q u a l l y important i n our commercial s e t t i n g was t h e o p p o r t u n i t y t o d e v e l o p a w e l l d e f i n e d r e s e a r c h g o a l a n d t o d e s i g n and, a s d e s c r i b e d h e r e , s u c c e s s f u l l y demonstrate a b i o l o g i c a l containment f e a t u r e .


Background and Biology B a c u l o v i r u s e s a r e a f a m i l y o f r o d - s h a p e d , DNA v i r u s e s f o u n d e x c l u s i v e l y w i t h i n t h e A r t h r o p o d s and c h i e f l y w i t h i n t h e l a r v a e , i . e . c a t e r p i l l a r s a n d worms, o f t h e e c o n o m i c a l l y i m p o r t a n t L e p i d o p t e r a ( F o r r e v i e w s t h a t c o v e r most o f t h e general p o i n t s r a i s e d herein see: l 2 3.). T h e i r a s s o c i a t i o n with n a t u r a l disease epidemics, o r e p i z o o t i c s , suggests t h e i r use as n a t u r a l i n s e c t i c i d e s . Nuclear p o l y h e d r o s i s v i r u s e s (NPVs) a r e t h e b a c u l o v i r u s g r o u p t h a t i s r e c e i v i n g t h e most i n t e r e s t as t a r g e t s f o r change by DNA engineering techniques. The n u c l e o c a p s i d s o f t h e s e v i r u s e s e x i s t i n two d i s t i n c t m o r p h o l o g i c a l f o r m s : a plasma membrane-budded, n o n - o c c l u d e d v i r u s (NOV), a n d an i n t r a n u c l e a r l y o c c l u d e d f o r m i n w h i c h many n u c l e o c a p s i d s are c o l l e c t e d together i n a p r o t e i n n e a r - c r y s t a l , t h e p o l y h e d r a l i n c l u s i o n body ( P I B ) . r

f

The n a t u r a l i n f e c t i o n c y c l e b e g i n s when a l e a f - f e e d i n g c a t e r p i l l a r i n g e s t s a PIB. A l k a l i n e c o n d i t i o n s i n t h e m i d g u t l e a d t o t h e d i s s o l u t i o n o f t h e PIB, r e l e a s i n g t h e n u c l e o c a p s i d s t o f u s e w i t h t h e midgut c e l l s a n d i n i t i a t e the i n f e c t i o n . Progeny v i r u s produced i n t h e s e i n i t i a l l y i n f e c t e d c e l l s a p p e a r t o be e x c l u s i v e l y NOVs, a c q u i r i n g a membrane a s t h e y e x i t t h r o u g h t h e p l a s m a membrane o f t h e cell. I t i s t h i s form o f t h e n u c l e o c a p s i d t h a t spreads t h e i n f e c t i o n throughout the l a r v a e . Subsequently i n f e c t e d t i s s u e s p r o d u c e a d d i t i o n a l NOV and a l s o p r o d u c e P I B s . A t the time o f death, t h e l a r v a e i s completely packed with PIBs. The model NPV s y s t e m i s an i s o l a t e f r o m t h e a l f a l f a l o o p e r , Autographa californica and, i n c o n j u n c t i o n w i t h L e p i d o p t e r a n c e l l c u l t u r e , makes an e x c e l l e n t l a b o r a t o r y system. I n c e l l c u l t u r e , t h e NOV i s ;he e x c l u s i v e i n f e c t i o u s form; PIBs p l a y no r o l e . A f t e r i n f e c t i o n , the n u c l e o c a p s i d makes i t s way t o t h e n u c l e u s , where r e p l i c a t i o n begins. The i n i t i a l s t e p s i n t h e v i r a l r e p l i c a t i o n c y c l e a r e p e r f o r m e d by c e l l u l a r f a c t o r s ,


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l e a d i n g t o a c a s c a d e o f v i r a l gene e x p r e s s i o n d r i v i n g t h e subsequent r e p l i c a t i o n s t e p s . From about 12 - 18 h o u r s p o s t - i n f e c t i o n , progeny n u c l e o c a p s i d s form i n t h e n u c l e u s . T h e s e b u d t h r o u g h c e l l u l a r membranes t o y i e l d e x t r a c e l l u l a r NOVs w h i c h s p r e a d t h e i n f e c t i o n t o o t h e r c e l l s i n t h e culture. In t h e s t a g e s p o s t - 1 8 h o u r s , p r o g e n y n u c l e o c a p s i d s do not l e a v e t h e n u c l e u s . A l a t e v i r a l gene p r o d u c t , t h e p r o t e i n p o l y h e d r i n , commences e x p r e s s i o n a s gene e x p r e s s i o n r e l a t e d t o t h e p r o d u c t i o n o f NOVs winds down. By c a 18 hours p o s t - i n f e c t i o n , p o l y h e d r i n begins t o c r y s t a l l i z e i n the c e l l nucleus. The n u c l e o c a p s i d s p r e s e n t h e r e a r e caught, o r occluded, i n t h e growing c r y s t a l , t h e PIB. T h e r e c a n be s e v e r a l h u n d r e d PIBs p e r n u c l e u s . This p r o c e s s c o n t i n u e s u n t i l t h e c e l l e v e n t u a l l y d i e s , c a 72 h r post-infection. The P I B s p r o d u c e d i n c u l t u r e a r e n o t infectious to cultured cells. They a r e p e r f e c t l y infectious to caterpillars. S e v e r a l f e a t u r e s o f b a c u l o v i r u s b i o l o g y have l e d t o an impressively r a p i d increase i n our understanding o f t h e i r r e p l i c a t i o n a t the molecular l e v e l . The n u c l e a r p o l y h e d r o s i s v i r u s e s grow i n c e l l c u l t u r e , and, a s d e s c r i b e d above, c a r r y o u t a l l known a s p e c t s o f t h e replication cycle. F u r t h e r m o r e , b a c u l o v i r u s e s have d o u b l e s t r a n d e d DNA genomes making them amenable t o a l l n u c l e i c a c i d t e c h n o l o g i e s a p p l i e d t o mammalian v i r a l s y s t e m s . F i n a l l y , t h e r e a r e t h e pragmatic f o r c e s d r i v i n g t h e use o f t h e v i r u s a s a p e s t i c i d e a n d as an e x p r e s s i o n v e c t o r f o r h e t e r o l o g o u s p r o t e i n s o f e x p e r i m e n t a l o r commercial interest. As m e n t i o n e d , NPVs d i r e c t t h e c e l l t o p r o d u c e l a r g e numbers o f P I B s . The P I B may be a s much a s 95% p o l y h e d r i n . The l e v e l o f s y n t h e s i s needed t o s u s t a i n t h i s l e a d s t o f a n t a s t i c amounts o f p o l y h e d r i n b e i n g p r o d u c e d i n l a t e stage i n f e c t e d c e l l s . E s t i m a t e s o f t h e amount o f p o l y h e d r i n p r o t e i n e x c e e d 25%. One i m p l i c a t i o n o f t h i s l a r g e amount o f m a t e r i a l i s t h a t a v e r y a c t i v e p r o m o t e r i s d r i v i n g t h e e x p r e s s i o n o f t h e p o l y h e d r i n gene, a n d i t i s now c l e a r t h a t v e r y l a r g e amounts o f p o l y h e d r i n RNA a r e produced i n these c e l l s . In such a case, a g e n e t i c e n g i n e e r w o u l d be i n c l i n e d t o t a k e a d v a n t a g e o f t h e p r o m o t e r and, l e v e r a g i n g i t w i t h o t h e r presumed u s e f u l


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f e a t u r e s o f t h e s y s t e m , employ i t i n an e x p r e s s i o n v e c t o r for h e t e r o l o g o u s genes i n s e r t e d under the c o n t r o l o f t h a t promoter. T h i s has b e e n t h e c a s e f o r t h e NPV p o l y h e d r i n promoter. The f e a t u r e s o f t h i s s y s t e m h a v e b e e n e l a b o r a t e d i n r e c e n t r e v i e w a r t i c l e s ( 4 . 5 , 6) . The o n l y r o l e f o r p o l y h e d r i n a p p e a r s t o be P I B f o r m a t i o n , w h i c h i s i r r e l e v a n t t o t h e p r o d u c t i o n o f in vitro i n f e c t i v e NOVs. Genes can t h e r e f o r e be i n s e r t e d i n p l a c e o f t h e p o l y h e d r i n gene, e l i m i n a t i n g i t s f u n c t i o n . The r e s u l t i n g v i r a l genome w i l l i n i t i a l l y d i r e c t t h e p r o d u c t i o n o f NOVs t h e n , w i t h t h e t i m e c o u r s e o f p o l y h e d r i n p r o t e i n gene e x p r e s s i o n , go o n t o p r o d u c e p r o d u c t as d i r e c t e d by t h e i n s e r t e d DNA. E x p e r i m e n t a l l y , DNA i s i n t r o d u c e d i n t o t h e v i r a l chromosome as f o l l o w s ( d e s c r i b e d i n d e t a i l w i t h e x a m p l e s i n 4, 6 ) : To f a c i l i t a t e w o r k i n g w i t h t h e p o l y h e d r i n g e n e , a s m a l l p o r t i o n o f t h e l a r g e v i r a l chromosome, c o n t a i n i n g t h e gene and i t s f l a n k i n g r e g i o n s , a r e c l o n e d i n t o a b a c t e r i a l plasmid. The p o l y h e d r i n c o d i n g r e g i o n s c a n (but do n o t have to) be removed i n the p r o c e s s o f i n t r o d u c i n g u s e f u l r e s t r i c t i o n enzyme s i t e s i n t h e v i c i n i t y o f t h e initiating ATG. O n c e c o n s t r u c t e d , f o r e i g n DNA c a n b e i n t r o d u c e d i n t o t h e p l a s m i d a t one o f t h e s e s i t e s , p l a c i n g i t downstream o f the presumptive promoter r e g i o n . As an a s i d e , t h e e x a c t n a t u r e o f the p o l y h e d r i n promoter and i t s a b i l i t y t o p r o d u c e l a r g e a m o u n t s o f RNA a r e o n l y now b e i n g e s t a b l i s h e d (2 a n d r e f e r e n c e s c o n t a i n e d t h e r e i n ) . It i s a fact that genes e x p r e s s e d from such c o n s t r u c t s produce f a r l e s s p r o t e i n than i s produced by the n a t u r a l p o l y h e d r i n gene. The r e a s o n s f o r t h i s d i s c r e p a n c y h a v e n o t b e e n f u l l y e l u c i d a t e d (see above r e f e r e n c e s f o r d i s c u s s i o n ) . Once c o n s t r u c t e d , t h e p l a s m i d s e q u e n c e s a r e i n t r o d u c e d i n t o t h e v i r u s chromosome b y c o - t r a n s f e c t i o n o f t h e p l a s m i d w i t h n a k e d w i l d t y p e v i r a l DNA. R e c o m b i n a t i o n o c c u r s a t a l o w b u t u s e f u l f r e q u e n c y b e t w e e n t h e s e DNAs a t t h e i r homologous r e g i o n s , f l a n k i n g the p o l y h e d r i n gene. The progeny v i r u s from the c o - t r a n s f e c t i o n are plaqued, and the plaques are scored for type. Under a d i s s e c t i n g m i c r o s c o p e , t h e r e c o m b i n a n t s can be s e l e c t e d from t h e w i l d t y p e s b a s e d on t h e i r d i s t i n c t i v e p l a q u e m o r p h o l o g y due t o a lack of PIBs. Once s e l e c t e d and a s t o c k p r e p a r e d , one has a v i r u s w h i c h on i n f e c t i o n o f c e l l s w i l l d i r e c t the p r o d u c t i o n o f b o t h a d d i t i o n a l v i r u s and the product o f the


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Baculoviruses

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In o u r p l a n n i n g f o r t h e m o d i f i c a t i o n o f an i n s e c t p a t h o g e n , we f o u n d t h a t NPVs met many o f t h e c r i t e r i a i n o u r m e n t a l check l i s t : t h e y have r e a s o n a b l e a c t i v i t y i n t h e i r u n a l t e r e d s t a t e , t h e y have a l r e a d y r e c e i v e d some c o m m e r c i a l use and, a s a r e s u l t , have e s t a b l i s h e d a good s a f e t y r e c o r d and r e g u l a t o r y h i s t o r y , t h e y c a n be m a n i p u l a t e d a t t h e m o l e c u l a r l e v e l , a n d we c o u l d d e v i s e a r e a l i s t i c s c e n a r i o f o r a l t e r a t i o n , w h i c h we a n t i c i p a t e w i l l i n c r e a s e t h e i r efficacy. L a s t l y , an u n a v o i d a b l e c o n c e r n i n t h i s a r e n a i s obtaining permission f o r the experimental f i e l d a p p l i c a t i o n of such a product. A very d e f i n i t e plus i n our s e l e c t i o n o f t h i s g r o u p was t h a t we c o u l d d e v i s e a b i o l o g i c a l c o n t a i n m e n t scheme t h a t would a c t u a l l y l e a d t o a l o s s o f t h e r e c o m b i n a n t v i r u s a s i t grew i n i t s h o s t s a n d p r e v e n t i t s unchecked growth. T h i s a p p r o a c h w i l l be d i s c u s s e d i n d e t a i l below. The c r i t i c a l p o i n t i n u s i n g t h e v i r u s a s an i n s e c t i c i d e r e l a t i v e t o i t s u s e a s an e x p r e s s i o n v e c t o r i s summarized as f o l l o w s : A f o r e i g n gene, from a n y s o u r c e , c a n be p l a c e d i n t o t h e b a c u l o v i r u s chromosome s o t h a t upon i n f e c t i o n o f a c a t e r p i l l a r c e l l a gene p r o d u c t , p e r h a p s n e v e r e x p r e s s e d , or never expressed a t t h i s time, o r never expressed i n t h i s amount, i s now e x p r e s s e d . One h a s t h e o p p o r t u n i t y t h e n t o i n t r o d u c e n o v e l gene p r o d u c t s i n t o a n a t u r a l p o p u l a t i o n o f c a t e r p i l l a r s v i a t h e v i r u s a n d augment t h e i r c o n t r o l o v e r and above t h a t o f t h e v i r u s a l o n e a n d p e r h a p s i n ways t h a t c a n n o t be s u p p l a n t e d b y s t a n d a r d a g r i c u l t u r a l c h e m i c a l s . T h i s makes f o r some v e r y e x c i t i n g p o s s i b i l i t i e s . B a c u l o v i r u s e s have been c o m m e r c i a l l y l i m i t e d b e c a u s e o f shortcomings r e l a t i v e t o t h e i r chemical competition: Any one b a c u l o v i r u s i s a c t i v e a g a i n s t o n l y a l i m i t e d number o f



410


c a t e r p i l l a r s p e c i e s , w h i c h a t one p o i n t i n t i m e was a f e a t u r e t h o u g h t t o be an a s s e t . However, i n a c o n t r o l s i t u a t i o n , t h e r e can be c a t e r p i l l a r s p r e s e n t w h i c h a r e o u t s i d e t h e h o s t r a n g e , c o m p l i c a t i n g t h e usage d e c i s i o n . C a t e r p i l l a r s w h i c h a r e s u s c e p t i b l e t a k e a number o f days t o d i e a f t e r e a t i n g a l e t h a l dose, and l a t e r i n s t a r s can be quite refractory to infection. Baculoviruses are r a t h e r e n v i r o n m e n t a l l y u n s t a b l e , as w e l l . They a r e p a r t i c u l a r l y s e n s i t i v e t o u l t r a v i o l e t l i g h t , which decreases t h e i r e f f e c t i v e l i f e t i m e on t h e l e a f s u r f a c e . To o b t a i n commercial q u a n t i t i e s of m a t e r i a l , b a c u l o v i r u s e s must be p r o d u c e d i n l a r v a e , w h i c h i s p e r c e i v e d as a cumbersome retro-technology. This perception i s in fact a misconception. W h i l e c e r t a i n l y n o t a s t a n d a r d way o f p r o d u c i n g an i n s e c t i c i d e , i t i s a t e c h n i c a l l y demanding but very workable p r o c e s s . B e f o r e e l a b o r a t i n g on t h e ways i n which g e n e t i c e n g i n e e r i n g can a l l e v i a t e some o f t h e s e p r o b l e m s , i t i s w o r t h r e s t a t i n g some o f t h e well-known s a f e t y c h a r a c t e r i s t i c s o f u n a l t e r e d b a c u l o v i r u s e s , a l l o f w h i c h we a n t i c i p a t e w i l l be m a i n t a i n e d w i t h an e n g i n e e r e d v i r u s . B a c u l o — meaning r o d - s h a p e d — v i r u s e s have no known counterparts i n v e r t e b r a t e v i r u s e s , yet they are widely d i s t r i b u t e d i n nature. During e p i z o o t i c s i n s u s c e p t i b l e L e p i d o p t e r a , f o r example t h e g y p s y moth, t h e y a r e p r e s e n t i n u n b e l i e v a b l y h i g h numbers and d e n s i t i e s w i t h no a d v e r s e environmental e f f e c t s observed other than t o the host larvae. The a n i m a l s a f e t y t e s t i n g o f b a c u l o v i r u s e s has been v e r y extensive. S i m p l y summarized, t h e r e have been no u n e x p e c t e d a d v e r s e e f f e c t s on any v e r t e b r a t e and no e f f e c t s on any i n v e r t e b r a t e w h i c h i s not p a r t o f t h e h o s t r a n g e ( f o r r e v i e w see ^ ) . B a c u l o v i r u s e s have no p h y t o t o x i c i t y . S e v e r a l n a t u r a l l y - o c c u r r i n g b a c u l o v i r u s e s have now b e e n r e g i s t e r e d i n t h e U n i t e d S t a t e s f o r use as i n s e c t i c i d e s on f o r e s t r y and f o o d c r o p s . The c u r r e n t r e g u l a t o r y framework w i l l subject the f i r s t engineered b a c u l o v i r u s p e s t i c i d e to a h i g h l e v e l o f s c r u t i n y . Q u e s t i o n s o f t o x i c i t y a s i d e (as t h i s w i l l be h a n d l e d on a c a s e - b y - c a s e b a s i s ) , an i s s u e o f g e n e r a l and p r i m e c o n c e r n w i l l be t h e g e n e t i c s t a b i l i t y o f altered viruses. Going hand-in-hand with a rod-shaped n u c l e o c a p s i d i s t h e a b i l i t y o f t h e v i r u s t o accommodate



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g a i n s o r l o s s e s o f DNA b y s i m p l y e x p a n d i n g o r s h r i n k i n g t h e particle. F o r example, i n n a t u r a l p o p u l a t i o n s , b a c u l o v i r u s e s s e e m t o b e p r o n e t o a c q u i r i n g h o s t DNA sequences, which i n s e v e r a l cases examined appear t o be t r a n s p o s a b l e elements ( f o r r e v i e w see 1 & ) . With t h i s i n mind, the f o l l o w i n g questions are o f relevance t o the f i e l d testing of genetically altered viruses: To what e x t e n t do i n t r o d u c e d sequences change t h e o v e r a l l dynamics o f t h e genome a n d a r e t h e i n t r o d u c e d s e q u e n c e s , i n p a r t i c u l a r , more l i k e l y t o b e i n v o l v e d i n u n t o w a r d e v e n t s ? Having d e a l t e x t e n s i v e l y w i t h e n g i n e e r e d v i r u s e s b o t h in vivo a n d in vitro, we h a v e o b s e r v e d n o u n e x p e c t e d i n s t a b i l i t y o f t h e genome o v e r e i t h e r t h e e n t i r e c h r o m o s o m e o r t h e i n s e r t e d gene. N e i t h e r h a v e we d e t e c t e d a n y u n u s u a l r e c o m b i n a t i o n events i n v o l v i n g engineered v i r u s e s . An a d d i t i o n a l s a f e t y f e a t u r e o f t h e e n g i n e e r e d p r o d u c t we a r e developing i s a b i o l o g i c a l containment feature which w i l l m i n i m i z e t h e s p r e a d , b y growth, o f t h e p r o d u c t from t h e a p p l i c a t i o n s i t e , a n d i n t h i s s e n s e , make t h e a l t e r e d v i r u s more " s a f e " t h a n t h e w i l d t y p e . T h e r e a r e a number o f t e c h n i c a l means u n d e r consideration f o r a l l e v i a t i n g the shortcomings o f b a c u l o v i r u s e s as i n s e c t i c i d e s . Of the s e v e r a l problems, p e r h a p s t h e most r e a d i l y a p p r o a c h a b l e t h r o u g h e n g i n e e r i n g i s improving the speed o f k i l l . S h o r t e n i n g t h i s t o as l i t t l e a s o n e d a y w o u l d make a r a d i c a l d i f f e r e n c e i n t h e commercial u t i l i t y and acceptance o f these agents. It i s c u r r e n t l y assumed t h a t i n f e c t e d l a r v a e d i e because t h e y a r e overwhelmed by t h e course o f the i n f e c t i o n , and i t takes s e v e r a l days f o r t h i s t o o c c u r . However, employing t h e v i r u s a s a n e x p r e s s i o n v e c t o r t o d e l i v e r some d e l e t e r i o u s gene p r o d u c t i n t o t h e l a r v a e c o u l d g r e a t l y i n c r e a s e t h e speed o f k i l l . I t s h o u l d be n o t e d t h a t J i i l l as used h e r e r e a l l y refers to a disabling or i n a c t i v a t i o n o f the larvae such t h a t feeding ceases and, i d e a l l y , a farmer can v i s u a l l y a s s e s s t h e c o n t r o l he i s a c h i e v i n g . Most genes chosen f o r i n t r o d u c t i o n w i l l code f o r a p r o t e i n t h a t f a l l s i n t o any o f s e v e r a l d i f f e r i n g c l a s s e s which might f u l f i l l these c r i t e r i a . T h i s concept has been e l a b o r a t e d i n l l 1 2 . T h e r e a r e a number o f p r o t e i n a c e o u s t o x i n s produced i n l i v i n g systems. Arthropods produce a great variety; however, these are g e n e r a l l y q u i t e p o o r l y r



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c h a r a c t e r i z e d ( f o r r e v i e w s see 13. 14). Perhaps the best known and most c h a r a c t e r i z e d c a t e r p i l l a r - s p e c i f i c t o x i n i s t h a t p r o d u c e d by t h e s e v e r a l v a r i e t i e s o f Bacillus thuringiensis. A d d i t i o n a l l y , t h e r e a r e a number o f enzymes, s u c h as p r o t e a s e s and c h i t i n a s e s , as w e l l as e n z y m a t i c i n h i b i t o r s w h i c h might be examined. E x c i t i n g to c o n t e m p l a t e a r e i n s e c t neurohormones. T h e i r expression at h i g h l e v e l s c o u l d l e a d t o p r o v o c a t i v e phenomenon w i t h i n t h e l a r v a e and p e r h a p s w i t h i n t h e p e s t p o p u l a t i o n . In t h i n k i n g t h r o u g h a p l a n f o r m o d i f y i n g t h e v i r u s by gene i n s e r t i o n , i t must be k e p t i n mind t h a t t h e i d e a l s e l e c t i o n would not have a c t i v i t y much o u t s i d e t h e t a r g e t r a n g e . Furthermore, i t s h o u l d be c o m p a t i b l e w i t h p r o d u c i n g t h e v i r u s p r o d u c t commercially i n larvae. This loose grouping of candidate p r o t e i n s w i l l h e r e a f t e r be r e f e r r e d t o as t o x i n s . I t i s i r o n i c t h a t t h e i n i t i a l f o c u s on b a c u l o v i r u s e s as i n s e c t i c i d e s stemmed f r o m t h e i r h i g h d e g r e e o f s p e c i f i c i t y f o r a l i m i t e d number o f L e p i d o p t e r a n l a r v a e and c o n s e q u e n t lessened environmental concern. T h i s has i n p a r t b e e n t h e i r u n d o i n g and mechanisms a r e now b e i n g c o n t e m p l a t e d f o r e n l a r g i n g the host range. As a d o p t e d i n b a c u l o v i r o l o g y , t h e c o n c e p t o f h o s t r a n g e t r e a t s a p e r m i s s i v e i n f e c t i o n as one i n w h i c h t h e l a r v a e i s f u l l y s u s c e p t i b l e t o t h e v i r u s , l e a d i n g i n t h e c a s e o f NPVs t o t h e p r o d u c t i o n o f P I B s and ending i n the death of the l a r v a e . W i t h i n t h i s , i t i s now a p p r e c i a t e d t h a t t h e r e a r e a l s o a wide r a n g e and p r o b a b l y h i g h f r e q u e n c y o f s e m i - p e r m i s s i v e i n f e c t i o n s i n w h i c h some a s p e c t o f t h e v i r a l l i f e c y c l e (as measured a g a i n s t some r e f e r e n c e ) i s c u r t a i l e d . There are a l s o non-permissive infections. T h e r e a r e two a p p r o a c h e s t o i m p r o v i n g t h e h o s t range o f baculoviruses. There i s the c o n f r o n t a t i o n a l approach of c l e a r l y d e l i n e a t i n g i n some p a r t i c u l a r c a s e o f v i r u s t o h o s t where t h e h o s t r a n g e r e s t r i c t i o n ( s ) l i e s . This i s l i k e l y t o be a c o n s i d e r a b l e u n d e r t a k i n g f o r even j u s t one case. The p r o b l e m w i t h t h i s a p p r o a c h as i t r e l a t e s t o d e v e l o p i n g a b e t t e r p e s t i c i d e i s t h a t t h e answer i s l i k e l y t o be c a s e s p e c i f i c ; t h a t i s , the d e f i c i e n c y i n a v i r u s ' i n t e r a c t i o n w i t h a p a r t i c u l a r h o s t w i l l be u n i q u e and knowing t h e answer w i l l n o t t e l l us how t o make t h a t p a r t i c u l a r v i r u s i n t e r a c t b e t t e r w i t h the next d e s i r a b l e t a r g e t t h a t i s not p a r t of i t s host range.



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A b e t t e r a p p r o a c h would be t o a c t u a l l y i g n o r e t h e s p e c i f i c s o f host range d e t e r m i n a t i o n and i n s t e a d d e v e l o p a method t h a t a l l o w s a n e n d - r u n a r o u n d v i r a l - h o s t i n t e r a c t i o n d e f i c i e n c i e s and p e r m i t s t h e v i r u s t o ( i f p e r h a p s n o t c a r r y out a f u l l r e p l i c a t i o n c y c l e ) a c t a s a n e x p r e s s i o n v e c t o r and d e l i v e r t o t h e h o s t a t o x i n gene t h a t w i l l be e x p r e s s e d and d i s p a t c h t h e p e s t . The i d e a h e r e i s t h a t t h e c o r r e c t p r o m o t e r d r i v i n g t h e e x p r e s s i o n o f a t o x i n gene d u r i n g a weak o r i n a p p a r e n t i n f e c t i o n o r p e r h a p s no r e p l i c a t i o n a t a l l would n o n e t h e l e s s a l l o w s u f f i c i e n t e x p r e s s i o n o f t h e gene t o a c h i e v e t h e d e s i r e d r e s u l t . An example o f how t h i s might work has been e l e g a n t l y d e m o n s t r a t e d b y L o i s M i l l e r ' s l a b o r a t o r y (15 1 6 ) . By e m p l o y i n g t h e t e c h n o l o g y d e s c r i b e d above, C a r b o n e l l e t . a l . c o n s t r u c t e d a n Autographa californica NPV w h i c h c o n t a i n e d two f o r e i g n genes, e a c h u n d e r t h e c o n t r o l o f a d i f f e r e n t p r o m o t e r (15.) . The p o l y h e d r i n p r o m o t e r was u s e d t o e x p r e s s a f u s i o n o f the N-terminal region o f the p o l y h e d r i n p r o t e i n t o E. coli p-galactosidase. The v i r u s a l s o c o n t a i n e d , a d j a c e n t l y located, the chloramphenicol a c e t y l t r a n s f e r a s e gene under t h e t r a n s c r i p t i o n a l c o n t r o l o f t h e l o n g t e r m i n a l r e p e a t (LTR) o f t h e Rous sarcoma v i r u s (RSV). The RSV LTR had p r e v i o u s l y been c h a r a c t e r i z e d a s a p r o m o t e r c a s s e t t e w h i c h would f u n c t i o n i n a wide v a r i e t y o f c e l l t y p e s , i n c l u d i n g Drosophila and mammalian c e l l l i n e s . I t was o f i n t e r e s t t o d e t e r m i n e i f t h i s arrangement would a l l o w e x p r e s s i o n o f e i t h e r o f t h e s e e a s i l y - a s s a y e d r e p o r t e r genes when t h e v i r u s was i n t r o d u c e d i n t o n o v e l h o s t s . r

When t h e v i r u s was u s e d t o i n f e c t a known, f u l l y p e r m i s s i v e c e l l l i n e f r o m Spodoptera frugiperda, both genes were e x p r e s s e d . CAT a c t i v i t y was d e t e c t e d e a r l y i n t h e i n f e c t i o n a t about s i x h o u r s , a n o t s u r p r i s i n g r e s u l t s i n c e t h e RSV LTR c o n t a i n s a n RNA p o l y m e r a s e I I p r o m o t e r and t h i s e n z y m a t i c a c t i v i t y , p r e - e x i s t i n g i n t h e c e l l , s h o u l d be amongst t h e f i r s t t o a c t on t h e v i r a l chromosome. Late i n the i n f e c t i o n , a t the time t h a t the p o l y h e d r i n p r o m o t e r i s known t o become a c t i v e , | 3 - g a l a c t o s i d a s e a c t i v i t y was d e t e c t e d . The NOV Drosophila h o s t range v i r u s were

f o r m o f t h e v i r u s was a l s o u s e d t o i n f e c t a c e l l l i n e in vitro. F l i e s a r e not p a r t o f t h e o f t h i s p a r t i c u l a r b a c u l o v i r u s and no p r o g e n y a n t i c i p a t e d . The s i t u a t i o n w i t h r e g a r d t o gene


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e x p r e s s i o n as d r i v e n by t h e s e two v e r y d i f f e r e n t p r o m o t e r s was n o t known. As i t t u r n e d o u t , s t a r t i n g a t about 12 h o u r s p o s t - a p p l i c a t i o n , CAT a c t i v i t y was d e t e c t e d and c o n t i n u e d t o r i s e f o r a number o f h o u r s . Polyhedrin p r o m o t e r a c t i v i t y , as r e p o r t e d by P - g a l a c t o s i d a s e , was never detected. The a u t h o r s were a b l e t o d e t e c t what t h e y f e l t was a v e r y low l e v e l o f DNA r e p l i c a t i o n i n Drosophila. The i m p o r t a n t p o i n t i s t h a t t h e d e t e c t i o n o f CAT a c t i v i t y i n Drosophila demonstrates t h a t the v i r u s i s e n t e r i n g the c e l l s and i s b e i n g u n c o a t e d t o f r e e t h e genome f o r transcriptional activity. The p r e s u m p t i v e r e a s o n f o r l a c k o f P - g a l a c t o s i d a s e a c t i v i t y i s the absence o f c e l l u l a r or v i r a l products necessary f o r p o l y h e d r i n promoter a c t i v i t y . A l o g i c a l e x p e r i m e n t a l e x t e n s i o n o f t h i s work i s a l s o p r e s e n t e d by t h e s e a u t h o r s . The v i r u s , as NOVs, i s a d m i n i s t e r e d t o m o s q u i t o a d u l t s by (of a l l t h i n g s ) enema. The a u t h o r s were a b l e t o d e t e c t CAT a c t i v i t y i n m o s q u i t o midgut. T h i s i l l u s t r a t e s t h e p o i n t r a i s e d above: I t may be p o s s i b l e t o e f f e c t i v e l y i n c r e a s e t h e h o s t r a n g e o f t h e v i r u s by e m p l o y i n g i t as a d e l i v e r y v e h i c l e f o r a t o x i n gene. P r e s u m a b l y , had t h e CAT gene been r e p l a c e d by a gene whose p r o d u c t was t o x i c t o m o s q u i t o e s , i t w o u l d have " k i l l e d " the mosquito a d u l t s . T h i s k i n d o f c o n s t r u c t w i l l be v e r y u s e f u l i n determining the s a f e t y of engineered v i r u s e s t o non-target organisms. When t h e two-gene v i r u s i s a p p l i e d t o mouse c e l l s , no a c t i v i t y i s d e t e c t e d from e i t h e r p r o m o t e r (1£) . S i n c e i t i s known t h a t t h e RSV LTR i s a c t i v e i n mouse c e l l s , the l a c k o f a c t i v i t y i s thought t o i n d i c a t e t h a t the v i r u s i s not b e i n g uncoated i n s i d e these c e l l s . This very i m p o r t a n t o b s e r v a t i o n had an i m m e d i a t e l y p o s i t i v e e f f e c t on our thoughts r e g a r d i n g the s a f e t y o f b a c u l o v i r a l pesticides. I t a l s o p r o v i d e s some g u i d a n c e i n d e s i g n i n g v i r a l m o d i f i c a t i o n s . Given the p o t e n t i a l f o r a c t i v i t y i n n o n - L e p i d o p t e r a n h o s t s , one would i d e a l l y c h o o s e a t o x i n gene t h a t was as L e p i d o p t e r a n - s p e c i f i c as p o s s i b l e and a mode o f e x p r e s s i n g t h i s gene, w h i c h i f a t a l l p o s s i b l e , a d d e d a n o t h e r l a y e r o f s a f e t y . These o p t i o n s e x i s t and i n c o n j u n c t i o n with our b i o l o g i c a l containment f e a t u r e ( d e s c r i b e d below) have a l l o w e d us t o become c o m f o r t a b l e


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w i t h t h e i d e a o f d e v e l o p i n g and f i e l d t e s t i n g a g e n e t i c a l l y modified baculovirus insecticide. O t h e r o p i n i o n s do e n t e r i n t o s u c h a d e c i s i o n . The E n v i r o n m e n t a l P r o t e c t i o n Agency h a s j u r i s d i c t i o n o v e r t h e f i e l d testing of pesticides. T h e i r most r e c e n t p o l i c y statement i n d i c a t e s t h a t a l l g e n e t i c a l l y m o d i f i e d organisms w i l l be s u b j e c t t o r e v i e w b e f o r e any f i e l d t e s t i n g b e g i n s (12). M o d i f i c a t i o n s such as those i m p l i c i t h e r e that i s , t h e a d d i t i o n o f n o n - v i r a l DNA t o t h e v i r u s chromosome, w i l l g e t t h e maximum l e v e l o f s c r u t i n y . L i k e l y t o be o f p a r t i c u l a r i n t e r e s t a r e questions o f environmental p e r s i s t e n c e , genomic s t a b i l i t y a n d s a f e t y o f t h e s p e c i f i c construct t o non-target organisms. What c a n b e s a i d about t h e e c o l o g y o f b a c u l o v i r u s e s ? As with a l l v i r u s e s , they a r e o b l i g a t e c e l l u l a r p a r a s i t e s , r e l y i n g on t h e i r h o s t s f o r r e p l i c a t i o n m a c h i n e r y . T h i s has some i m p o r t a n t i m p l i c a t i o n s f o r f i e l d m o n i t o r i n g a n d s h o u l d serve t o r e s t r i c t i n t e r e s t t o r e p l i c a t i o n p r o f i c i e n t v i r u s and v i r u s t h a t h a s t h e o p p o r t u n i t y t o g e t i n t o a h o s t . (See 1 £ f o r a t h o r o u g h d i s c u s s i o n o f t h e s e i s s u e s . ) . A f a c t o r such as s u n l i g h t w i l l i n a c t i v a t e t h e great m a j o r i t y o f a p p l i e d v i r u s o v e r a s h o r t p e r i o d o f t i m e . The i n a c t i v a t e d v i r u s , w h i l e p e r h a p s d e t e c t a b l e b y some a n t i g e n - b a s e d a s s a y , i s o f no i n t e r e s t i n terms o f s a f e t y . To a s s e s s t h e l e v e l o f a c t i v e v i r u s w i l l r e q u i r e d i r e c t assay i n t h e i r host, c a t e r p i l l a r s . These a s s a y s a r e v e r y s e n s i t i v e because l i v e v i r u s i s very i n f e c t i v e . Many w o r k e r s have n o t e d t h e s e n s i t i v i t y o f b a c u l o v i r u s e s t o t h e u l t r a v i o l e t l i g h t component o f s u n l i g h t ( F o r a recent review o f a l l aspects o f v i r u s environmental s t a b i l i t y s e e 12..) . U n f o r m u l a t e d a n d u n p r o t e c t e d v i r u s a p p l i e d t o a c r o p l o s e s much o f i t s b i o l o g i c a l a c t i v i t y w i t h r a t e s a p p r o a c h i n g 50% a day. T h i s i s p r e s u m a b l y due t o genomic damage. V i r u s w h i c h s u r v i v e s t h i s a n d makes i t s way t o t h e s o i l h a s a much b e t t e r c h a n c e . Baculoviruses, p e r h a p s due t o t h e i r p o l y h e d r i n c o v e r i n g , a r e v e r y l o n g l i v e d when s e q u e s t e r e d i n t h e s o i l , w i t h a t i m e frame measured i n y e a r s . I t i s c u r r e n t l y thought t h a t t h i s r e s e r v o i r p r o v i d e s t h e i n o c u l u m f o r most o f t h e n a t u r a l l y induced v i r a l e p i z o o t i c s . The s t a b i l i t y o f t h e v i r u s i n s o i l i s s o m e t h i n g t h a t must be a n t i c i p a t e d a n d a c c e p t e d b y both t h e p e s t i c i d e developer and t h e r e g u l a t o r y a g e n c i e s .


f



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A g e n e t i c a l l y engineered v i r u s applied to a f i e l d w i l l s t i l l be d e t e c t a b l e s e v e r a l y e a r s l a t e r . As p a r t o f o u r development e f f o r t , we have c o n d u c t e d a s u r v e y o f t h e s t a b i l i t y o f t h e v i r a l genome. The c l e a r summation o f t h i s work i s t h a t an e n g i n e e r e d v i r u s i s no l e s s s t a b l e t h a n a w i l d - t y p e v i r u s , when examined b o t h o v e r t h e e n t i r e chromosome and when r e s t r i c t e d t o t h e a l t e r e d portion. F o r example, we have c o n s t r u c t s i n w h i c h we have u t i l i z e d v a r i o u s LTRs as p r o m o t e r s f o r ( 5 - g a l a c t o s i d a s e i n p l a c e o f t h e p o l y h e d r i n p r o m o t e r and gene. In a n o t h e r c a s e , we have l e f t t h e p o l y h e d r i n p r o m o t e r as i s b u t r e p l a c e d t h e gene w i t h P - g a l a c t o s i d a s e . N e i t h e r o f t h e s e v i r u s t y p e s i s c a p a b l e o f p r o d u c i n g PIBs and must t h e r e f o r e be p a s s a g e d i n c e l l c u l t u r e o r i n c a t e r p i l l a r s by injection. T h i s has been done and many p r o g e n y p l a q u e s have been s c r e e n e d f o r t h e p a r e n t a l a b i l i t y t o c o n v e r t t h e i n d i c a t o r x - g a l t o i t s b l u e form. We have n e v e r o b s e r v e d a d e f i c i e n t plaque. We have a l s o t r i e d t o f o r c e t h e v i r u s t o undergo u n u s u a l recombination events. In t h i s s e t o f e x p e r i m e n t s , c u l t u r e d c e l l s were s i m u l t a n e o u s l y i n f e c t e d w i t h two v i r u s t y p e s . One, w i l d t y p e v i r u s (PIB +, c l e a r p l a q u e ) and, two, a recombinant i n which the p o l y h e d r i n promoter i s d r i v i n g P - g a l a c t o s i d a s e e x p r e s s i o n (PIB -, b l u e p l a q u e ) . The p r o g e n y o f t h i s i n f e c t i o n were p l a q u e d and s c r e e n e d f o r any t h a t were p o t e n t i a l r e c o m b i n a n t s (PIB +, b l u e p l a q u e ) . In f a c t , we have c o n s i s t e n t l y f o u n d q u i t e a h i g h p e r c e n t a g e o f these. T h i s work needs f u r t h e r development, b u t i t i s o u r f e e l i n g b a s e d on t h e a n a l y s i s o f a number o f t h e s e p l a q u e s t h a t they are not n o v e l recombinants. R a t h e r , we t h i n k they are e i t h e r : genomic c o i n t e g r a t e s ( a n t i c i p a t e d t o be u n s t a b l e ) , n u c l e o c a p s i d s w i t h m u l t i p l e i n d e p e n d e n t genomes e n c a p s i d a t e d , o r n u c l e o c a p s i d s w h i c h have a g g r e g a t e d and c o n s e q u e n t l y y i e l d an impure p l a q u e t h a t i n i t i a l l y a p p e a r s t o be p u r e . None o f o u r a n a l y z e d p l a q u e s y i e l d e d a p u r e recombinant phenotype a f t e r s e v e r a l r e p l a q u i n g s , a r g u i n g a g a i n s t s t a b l e n o v e l recombinants but c o n s i s t e n t w i t h the above i n t e r p r e t a t i o n s . B i o l o g i c a l Containment The s a f e t y h i s t o r y o f none n g i n e e r e d b a c u l o v i r u s e s and t h e n a t u r e o f t h e a n t i c i p a t e d



31.

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m o d i f i c a t i o n work l e a d s us t o f e e l c o n f i d e n t a b o u t t h e safety o f our eventual product. R e c e n t h i s t o r y h a s shown t h a t t h e r e w i l l be t h o s e who a r e n o t s o s a n g u i n e . I n an e f f o r t t o i n c r e a s e t h e i r c o m f o r t l e v e l we have d e v i s e d a n d t e s t e d a b i o l o g i c a l c o n t a i n m e n t scheme f o r a m o d i f i e d virus. Our scheme t a k e s i n t o a c c o u n t t h e f o l l o w i n g : t h e e n g i n e e r e d v i r u s , h a v i n g h a d i t s p o l y h e d r i n gene r e p l a c e d by a t o x i n gene, w i l l i n g e n e r a l n o t be a b l e t o p r o d u c e PIBs , PIBs a r e d e s i r a b l e f o r p r o d u c t i o n , f o r m u l a t i o n , a n d a p p l i c a t i o n p u r p o s e s , t h e v i r u s i s v e r y s t a b l e once r e s e r v o i r e d i n t h e s o i l , a n d t h e v i r u s r e q u i r e s an i n s e c t c e l l i n w h i c h t o grow. To i n i t i a t e t h e scheme, c e l l s i n c u l t u r e a r e s i m u l t a n e o u s l y i n f e c t e d with a w i l d type v i r u s and t h e recombinant, p o l y h e d r i n d e f i c i e n t v i r u s . Inside the c e l l n u c l e u s , an i n f e c t i o n i s e s t a b l i s h e d b y b o t h v i r u s t y p e s . The r e c o m b i n a n t i s a s r e p l i c a t i o n p r o f i c i e n t a s t h e w i l d type, except f o r i t s i n a b i l i t y t o produce PIBs. This f u n c t i o n i s c a r r i e d o u t by t h e w i l d t y p e . The PIBs t h a t form i n t h e n u c l e u s o c c l u d e n u c l e o c a p s i d s o f b o t h t h e w i l d t y p e and t h e r e c o m b i n a n t v i r u s , a s t h e y a r e i n d i s t i n g u i s h a b l e a t t h i s p o i n t . We r e f e r t o s u c h PIBs a s

mixed-composition

PIBs

(MC-PIB).

The m u l t i t u d e o f PIBs p r o d u c e d d u r i n g t h i s p r o c e s s a r e composed o f b o t h t y p e s o f i n p u t n u c l e o c a p s i d s . These p r o g e n y PIBs c a n be f e d t o c a t e r p i l l a r s , i n i t i a t i n g an i n f e c t i o n o f both v i r u s types. The n a t u r e o f t h e g r o w t h dynamics between t h e v i r u s t y p e s c a n be f o l l o w e d i n two ways. The PIBs t h e m s e l v e s c a n be d i s s o l v e d a n d t h e i r n u c l e o c a p s i d components a n a l y z e d b y DNA r e s t r i c t i o n enzyme d i g e s t i o n and subsequent f i l t e r h y b r i d i z a t i o n a n a l y s i s . T h i s a l l o w s an a s s e s s m e n t o f t h e p r o p o r t i o n s o f w i l d t y p e t o r e c o m b i n a n t v i r u s i n t h e MC-PIB. A l s o , t h e f r e e NOVs p r e s e n t i n an i n f e c t e d c a t e r p i l l a r c a n be c o l l e c t e d a n d analyzed by plaquing i n c e l l c u l t u r e . T h i s p r o v i d e s an assessment o f t h e frequency o f w i l d type t o recombinant v i r u s free i n the host. The c o m b i n a t i o n o f t h e s e two t e c h n i q u e s a l l o w s one t o f o l l o w t h e f a t e o f a recombinant v i r u s as i t i s passaged v i a t h e MC-PIB t e c h n i q u e t h r o u g h s u c c e e d i n g c a t e r p i l l a r s . We have now gone t h r o u g h t h e c r e a t i o n a n d t r a c k i n g o f s e v e r a l d i f f e r e n t MC-PIBs, a n d i t i s c l e a r f r o m t h i s t h a t


418



the technique contributes t o the s o l u t i o n o f several problems. F i r s t , i t allows the natural delivery of a recombinant, polyhedrin d e f i c i e n t v i r u s t o a pest population i n the field. Second, i t p r o v i d e s an economical means f o r mass p r o d u c i n g a r e c o m b i n a n t v i r u s i n a g r i c u l t u r a l l y r e l e v a n t amounts b y a l l o w i n g t h e p r o d u c t i o n to proceed v i a c a t e r p i l l a r s infected through t h e i r d i e t . F i n a l l y , i t p r o v i d e s a means o f b i o l o g i c a l c o n t a i n m e n t . I t i s o u r o b s e r v a t i o n t h a t i f an MC-PIB o f g i v e n r a t i o of w i l d type t o recombinant v i r u s i s f e d t o a c a t e r p i l l a r , and t h e progeny PIBs a r e f e d t o another c a t e r p i l l a r and so on, each g e n e r a t i o n w i l l see the frequency o f recombinant v i r u s decrease. A t some p o i n t s e v e r a l p a s s a g e s h e n c e , t h e r e c o m b i n a n t v i r u s h a s b e e n d i l u t e d o u t b y t h e more appropriate growth o f the w i l d type v i r u s . At this point, t h e recombinant v i r u s has e s s e n t i a l l y grown i t s e l f o u t o f e x i s t e n c e and has been w h o l l y r e p l a c e d b y t h e w i l d t y p e i n the PIBs.

H

10 :

*

0

1

2 Passage

3

4

1

10 : 10

5

Number

F i g u r e 1. The r a t i o o f recombinant t o w i l d - t y p e v i r u s changes w i t h each g e n e r a t i o n u n t i l o n l y t h e w i l d type remains.

The number of passages the recombinant virus survives is influenced by the initial ratio of recombinant to wild type virus used to infect the tissue culture cells. This is illustrated in Figure 1. Cells in culture were infected with either a multiplicity of infection of 10:1 or 10:10, recombinant: wild type, plaque forming units.



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Passage 0 i s t h e percentage o f recombinant plaques o b t a i n e d when t h e p r o g e n y v i r u s i n t h e c e l l c u l t u r e f l a s k were plaqued. Passage 1 r e f e r s t o t h e percentage o b t a i n e d from t h e f i r s t c a t e r p i l l a r s (Heliothis virescens, fourth i n s t a r ) f e d PIBs from t h e t i s s u e c u l t u r e c e l l s . As c a n be seen, t h e recombinant p e r s i s t s a t h i g h e r p e r c e n t a g e s f o r a l o n g e r p e r i o d o f t i m e when t h e i n i t i a l MC-PIB c o m p o s i t i o n i s skewed t o w a r d s t h e r e c o m b i n a n t . In t h i s example, t h e c a t e r p i l l a r s were i n f e c t e d w i t h t h e v i r u s b y p l a c i n g 1 0 MC-PIBs on t h e s u r f a c e o f t h e i r d i e t . I f t h i s i s r a i s e d t o 10^ MC-PIBs, t h e p e r s i s t e n c e o f t h e r e c o m b i n a n t i s e x t e n d e d o u t a t l e a s t one more g e n e r a t i o n . T h i s s e n s i t i v i t y t o i n o c u l u m l e v e l s i s p r o b a b l y due t o t h e r e q u i r e m e n t f o r a c e l l t o be c o i n c i d e n t a l l y i n f e c t e d w i t h b o t h a w i l d t y p e a n d a r e c o m b i n a n t v i r u s t o o b t a i n a n MCPIB. The l i k e l i h o o d o f t h i s o c c u r r i n g s h o u l d be s e n s i t i v e t o t h e i n i t i a l number o f v i r u s e s i n t r o d u c e d i n t o t h e caterpillars. 5

The MC-PIB phenomenon d e s c r i b e d p r o v i d e s b i o l o g i c a l c o n t a i n m e n t f o r an e n g i n e e r e d b a c u l o v i r u s . B a c u l o v i r u s e s r e q u i r e t h e p a c k a g i n g o f a PIB t o i n s u r e t h e i r s u r v i v a l i n n a t u r e between i n f e c t i o n s . The p r o v i s i o n o f P I B - p a c k a g i n g f o r a P I B - d e f i c i e n t r e c o m b i n a n t v i r u s by c o i n f e c t i o n w i t h a w i l d type v i r u s accomplishes t h i s . T h i s c o u l d have been counter t o t h e s a f e t y i s s u e , but f o r t u n a t e l y i n t h i s case, t h e p a c k a g i n g mechanism i s n o t f o o l p r o o f a n d w i t h s u c c e e d i n g i n f e c t i o n s , t h e r e c o m b i n a n t f i n d s i t s way l e s s f r e q u e n t l y i n t o t h e PIB and i s g r a d u a l l y l o s t . Whenever a MC-PIB h a r b o r e d i n t h e s o i l e x i t s v i a t h e i n f e c t i o n p r o c e s s , i t w i l l have c o n t r i b u t e d t o a d e c r e a s e i n t h e p r o p o r t i o n o f recombinants. This w i l l insure that the r e c o m b i n a n t does n o t s p r e a d t h r o u g h t h e e n v i r o n m e n t b y u n c h e c k e d growth a f t e r i t s a p p l i c a t i o n . In t h i s p a p e r , we have d i s c u s s e d b a c u l o v i r u s e s , i n s e c t p a t h o g e n s w h i c h we f e e l have g r e a t p o t e n t i a l a s i n s e c t c o n t r o l agents. The s h o r t c o m i n g s t h a t have been t h e i r p r o b l e m i n t h e p a s t may be e l i m i n a t e d by e m p l o y i n g v i r a l biology i n the service of genetic engineering technologies. We have p r e s e n t e d s c e n a r i o s i n w h i c h t h e i r e f f e c t i v e n e s s can be i m p r o v e d w h i l e t h e i r s a f e t y f e a t u r e s a r e m a i n t a i n e d and, i n a s e n s e , a c t u a l l y i m p r o v e d .


420


Acknowledgment s The author would like to thank Carol Ann Johnson, Pennina Safer, Peter LaPan, Lisa Hahn, and David Clemm for their invaluable contributions to the work described herein.


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Granados, R.R. and Federici, B.A. The Biology of Baculoviruses. vol 1 and 2: CRC Press: Boca Raton, FL, 1986. Maramorosch, K. and Sherman, K . E . V i r a l Insecticides for Biological Control: Academic: Orlando, F L . , 1985. Doerfler, W. and Bohm, P . , Eds. The Molecular Biology of Baculoviruses: Springer-Verlag: Berlin, 1986. Miller, D.W. et. al. In Genetic Engineering, vol 8: Setlow, J.K. and Hollaender, A . , Eds.; Plenum: New York, 1986; p. 277. Doerfler, W. In The Molecular Biology of Baculoviruses: Doerfler, W. and Bohm, P . , Eds.; Springer-Verlag: Berlin, 1986; p. 51. Luckow, V.A. and Summers, M.D. Biotechnol. 1988, 6, 47-55. Possee, R.D. and Howard, S.C. Nuc. Acids Res. 1987, 15, 10233-10248. Miyajima, A. et. a l . Gene 1987, 58, 273-281. Groner, A. In The Biology of Baculoviruses. vol 1: Granados, R.R. and Federici, B . A . , Eds.; CRC Press: Boca Raton, FL, 1986; Chapter 9. Miller, L . K . In The Biology of Baculoviruses, vol 1: Granados, R.R. and Federici, B . A . , Eds.; CRC Press: Boca Raton, FL, 1986; Chapter 11. Kirschbaum, J.B. In Ann. Rev. Entomol.; Mittler, T.E.,Ed.; Annual Reviews: Palo Alto, CA., 1985; p. 51. Keeley, L . L . and Hayes, T.K. Insect Biochem. 1987, 17, 639-651. Zlotkin, E . In Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol 10: Kerkut, G.A. and Gilbert, L.I., Eds.; Pergamon: New York, 1985; Chapter 15.



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14. Piek, T. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol 11; Kerkut, G.A. and Gilbert, L.I., Eds.; Pergamon: New York, 1985; Chapter 14. 15. Carbonell, L . F . et.al. J. V i r o l . 1985, 56, 153-160. 16. Carbonell, L . F . and Miller, L.K. Appl. Environ. Microbiol. 1987, 53, 1412-1417. 17. Federal Register, June 26, 1986. 18. Miller, D.W. e t . a l . In Prospects for Physical and Biological Containment of Genetically Engineered Organisms: G i l l e t t , J.W., E d . ; Ecosystems Research Center Report No. 114, Cornell Univ., 1987; Chapter 3. 19. Jacques, R.P. In Viral Insecticides for Biological Control: Maramorosch, K. and Sherman, K.E., Eds.; Academic: Orlando, FL., 1985; p. 285. RECEIVED

May 25, 1988


Biotechnology for Crop Protection - American Chemical Society

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